Biology subjects

Bobone, S.

Publications and source records attributed to Bobone, S..

4 recordsLinked to original sources

bioRxiv2021

Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin (HA), neuraminidase, matrix protein 2 (M2)) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and Number and Brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the case that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins. Statement of SignificanceInfluenza A virus (IAV) is a pathogen responsible for epidemics and occasional pandemics and, therefore, a significant burden on health systems. To develop innovative therapeutic approaches, a deeper understanding of the viral replication cycle is needed. For example, during the formation of new virions in infected cells, several viral components must assemble at the plasma membrane, but the molecular interactions involved in this process are not clearly understood. In this work, we use quantitative fluorescence microscopy methods to monitor the interplay between several viral proteins in live cell models. Our results underline the importance of the interactions between two specific proteins (M1 and M2) and shed light on the first steps in IAV assembly.

biophysics↗

bioRxiv2020

Peptide-based Inhibitors of Protein-Protein Interactions of Src Homology 2 Domain-Containing Phosphatase 2

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in multiple signaling pathways and a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective on disease-associated SHP2 mutants. Based on the consideration that pathogenic lesions cause signaling hyperactivation due to increased SHP2 association with cognate proteins, we developed peptide-based molecules with low nM affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation and an affinity for pathogenic variants of SHP2 up to 20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool to investigate the role of protein-protein interactions in the function of SHP2.

biophysics↗

bioRxiv2020

Inoculum effect of antimicrobial peptides

The activity of many antibiotics depends on the initial density of cells used in bacteria growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, since bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules to fight drug-resistant bacteria, since they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here we report the first systematic characterization of the inoculum effect for this class of antibacterial compounds. Thirteen peptides (including all-D enantiomers) and peptidomimetics were analyzed by measuring minimum inhibitory concentration values, covering more than 7 orders of magnitude in inoculated cell density. In all cases, we observed a significant inoculum effect for cell densities above 5 x 104 cells/mL, while the active concentrations remained constant (within the micromolar range) for lower densities. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause a killing effect. The observed effects question the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the inoculum dependence of the activity of antimicrobial peptides and peptidomimetics should be considered. Significance statementBacterial drug resistance is a crucial threat to global health and antimicrobials with novel mechanisms of action are severely needed. Antimicrobial peptides are natural molecules that kill bacteria mostly by perturbing their membranes and represent promising compounds to fight resistant microbes. Their activity is normally tested under standardized conditions of bacterial density. However, the bacterial load in clinically relevant infections varies by many orders of magnitude. Here we showed that the minimum peptide concentration needed for bacterial killing can vary by more than 100 times with an increase in the density of cells in the initial inoculum of the assay (inoculum effect) These findings question utility of the presently used activity screening assays and our current understanding of antimicrobial peptides.

biophysics↗

bioRxiv2018

Structural determinants of the interactions between Influenza A Virus Matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1-protein interactions and multimerization have not been clarified, yet.\n\nIn this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.

biophysics↗